American Journal of Agricultural and Biological Sciences 9 (3): 369-378, 2014 ISSN: 1557-4989 © 2014 C. Harris-Valle et al ., This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license doi:10.3844/ajabssp.2014.369.378 Published Online 9 (3) 2014 (http://www.thescipub.com/ajabs.toc)

VARIABILITY IN THE LIGNINOLYTIC ACTIVITY BY LENTINULA EDODES IN SUBMERGED CULTURE WITH AND GLUCOSE

1,4 Citlalli Harris-Valle, 2Elisa Valenzuela-Soto, 1Alfonso Sánchez, 3Rigoberto Gaitán-Hernández and 1Martín Esqueda

1Coordinación de Tecnología de Alimentos de Origen Vegetal, 2Coordinación de Ciencia de los Alimentos, Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Hermosillo, Sonora, México 3Instituto de Ecología, A.C., Xalapa, Veracruz, México 4Instituto Tecnológico Superior de Zacapoaxtla, Zacapoaxtla, Puebla, Mexico

Received 2014-01-03; Revised 2014-04-24; Accepted 2014-04-28 ABSTRACT The white-rot basidiomycete Lentinula edodes is the second most edible mushroom cultivated on the world. This fungus decomposes cell-wall associated macromolecules, is a natural degrader of lignin polymers. The differences in activities between strains of L. edodes provided useful information about the participation of enzymes in different development stages of the fungus. The effect of lignin on the fungal biomass production and activity behavior of ligninolytic enzymes when L. edodes is cultivated in a medium containing lignin with and without glucose as a carbohydrate source was tested. When glucose was present in the culture, lignin increase the mycelial biomass by 70% at 22 days compared to the control culture. The lignin media without glucose affected mycelial growth up to 20% less that the control without lignin and glucose. The activity of laccase, lignin , aryl alcohol oxidase, manganese dependent peroxidase and was modified depending on whether the medium had lignin and glucose, or lignin alone. A carbohydrate source is important to fungal growth, but the dissolution of lignin monomers might switch the signal that controls growth rate and enzymatic activity.

Keywords: Shiitake, Laccase, Lignin Peroxidase, Aryl Alcohol Oxidase, Manganese Dependent Peroxidase, Catalase

1. INTRODUCTION Lignin is a complex macromolecule that occurs in the cell walls of vascular plants. This polymer is considered Lentinula edodes (Berk) Pegler is considered an recalcitrant because it breaks down more slowly than alternative recycling agent for agricultural wastes and other wood polymers (hemicellulose, cellulose). there have been several studies to understand the rela- Nevertheless, there are some fungi species capable of tionship between its growth and ligninolytic activity. completely degrading lignin to carbon dioxide and L. edodes produces high amounts of and water (Elisashvili and Kachlishvili, 2009; Busse et al ., oxidases for bioconversion of lignocellulosic wastes. 2013). L. edodes is a white-rot fungus that can The differences in enzyme activities among strains of depolymerize lignin by means of an oxide-reduction L. edodes provided useful information about the mechanism called ligninolysis. This process occurs in participation of enzymes in different development two stages. First the subunit bonds are broken by Lignin stages (Silva et al ., 2005; Philippoussis et al ., 2011). Peroxidase (LiP) or (MnP) using Corresponding Author: Martín Esqueda, 1Coordinación de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Hermosillo, Sonora, México

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H2O2 as the oxidative . The same enzymes are from a stock plate (PDA) to submerged cultures (4 capable of producing H 2O2 from reductants such as mL) at 3.4 g in an orbital shaker (Lab-Line Glutathione (GSH) using O 2 as oxidant. During Instruments) at 25°C in the dark, using a 20 mL depolymerization oligomers of lignin and chemically cotton-plugged flask scintillation vials. stable phenolic monomers are produced (Aro et al ., The effect of lignin concentration in biomass 2005; Elisashvili and Kachlishvili, 2009). production after 12 days of L. edodes culture was During the second phase of ligninolysis, phenoxy assayed to a concentration of 0, 0.25, 0.5, 0.75, 1.0, 1.5 − radicals are converted into chemically stable products by and 2.0 mg mL 1. The highest biomass production was oxidases and oxido-reductases (v.g., Aryl Alcohol obtained by adding 1 mg of lignin/mL. Therefore the Oxidases (AAO) and laccase) (Guillén et al ., 2000). treatments were carried out without lignin and adding 1 Additionally, AAO can oxidize different alcohols, mg of alkali lignin/mL with Mw 28,000 (Aldrich No. producing Reactive Oxygen Species (ROS) that can be Cat. 37095-9) to media [glucose/peptone (40:10 w/v), used by to initiate the oxido-reduction pH 4.5] (LGP). Additionally, a liquid medium without mechanism as a cycle (Guillén and Evans, 1994). glucose (1 mg peptone/mL and 1 mg lignin/mL) (LP) Some authors have found that lignin derivatives and other with glucose and without lignin enhanced the mycelial growth in white-rot fungi, whereas lignin or compounds related to lignin subunits induce the (glucose/peptone (40:10 w/v), pH 4.5) (GP) were lignin degrading system (Beltrán-García et al ., 2001; prepared. The solution was sterilized at 120°C for 30 Ferrara et al ., 2002). However, the relationship between min prior to inoculation. The glucose was sterilized fungal growth and modification of activity of the separately to avoid the melanins formation. principal ligninolytic enzymes (LiP, MnP, AAO and 2.2. Fungal Biomass and Enzyme Extract laccase) when lignin polymer is added to culture medium is not clear. It has been proposed that lignin To determine biomass production, samples were is broken down when cultures of P. chrysosporium taken every 2 days, during 22 days, after 4 days of and L. edodes enter the stationary phase and nutrient incubation. The fungal biomass was harvested by levels are low (Ohga and Royse, 2001). filtration through Whatman No 1 filter, was washed with Nevertheless, in young cultures of L. edodes high 500 mL of distilled water and dried at 60°C for 24 h and ligninolytic activity has been observed to coincide with weighed. The extracellular proteins, contained in 3 mL the active degradation of carbohydrate sources are of aqueous residue culture, were concentrated by actively because the organisms require an alternative precipitation with acetone (1:5, v/v). The aqueous in order to continue breaking down the residue culture came from the same samples used for lignin (Leatham, 1985; Mata et al ., 1997). The paper biomass quantification. The solution was cooled at -20°C describes LiP, MnP, AAO and laccase production for 6 h and centrifuged (10 min at 2582 g in Sorvall profiles and induction in liquid cultures of L. edodes RC5S rotor SA-600) at room temperature (25°C). The by the presence of glucose and lignin in the growth pellet was suspended in 300 µL of 50 mM sodium medium to know about enzymatic modulation. The acetate and 10 mM KCl buffer, pH 4 (adding 250 M of hypothesis of this study is that lignin and glucose phenyl-methyl-sulfonyl-fluoride as a protease inhibitor). together supplemented in a liquid culture medium of The extracellular protein samples were kept at -80°C for L. edodes enhanced biomass and modified the activity enzymatic assays. Three replicates were prepared and pattern of ligninolytic enzymes. conducted in parallel. Independent cultures (3-6 flasks) were used for each replicate. The results are reported as 2. MATERIALS AND METHODS the mean with its standard deviation. 2.1. Culture Conditions 2.3. Enzyme Analyses Lentinula edodes commercial strain (IE-105) was Mn-dependent peroxidase (EC 1.11.1.13) activity provided from Fungi Strain Collection of the Institute was measured by monitoring the formation of Mn III- of Ecology (Xalapa, Mexico) (CS2: Original Code malonate complex at 270 nm during one minute in −1 −1 from Fungi Perfecti). The fungus was maintained on presence of 0.1 mM H 2O2 (ε270 = 11.59 M cm ), using Potato-Dextrose-Agar (PDA) (DIFCO) (20 g dextrose, 0.5 mM MnSO 4 and 50 mM sodium malonate (pH 4.5) 4 g potato infusion and 15 g agar per liter) at 4°C. (Faison and Kirk, 1985). Aryl alcohol oxidase (EC Mycelial inocu-lum (1 cm Ø; cut along the edge of a 1.1.3.7) was determined by the method of Guillén et al . 10-day-old actively growing colony) was transferred (1992) using 5 mM veratryl alcohol in 100 mM sodium

Science Publications 370 AJABS Citlalli Harris-Valle et al . / American Journal of Agricultural and Biological Sciences 9 (3): 369-378, 2014 phosphate buffer (pH 6.0) and lignin peroxidase (EC 40% higher than that of GP after 12 days of incubation). 1.11.1.14) activity assay mixture contained 25 mM However, the protein concentration in Lignin and Peptone sodium tartrate (pH 3.0), 2 mM veratryl alcohol and 0.1 (LP) was similar to that of GP from days 4 to 14 and then mM H 2O2 (Tien and Kirk, 1984). Both activities were decreased to lower levels (80% less than that of GP) measured by monitoring the increase in absorbance at compared to the other cultures ( Table 1 ). −1 −1 310 nm ( ε310 = 9300 M cm ). Laccase activity (EC 1.10.3.2) was estimated by 3.2. Enzymatic Activity −1 −1 monitoring at 420 nm ( ε =36 M cm ) (Boer et al ., 420 The activity of the four ligninolytic enzymes was 2004) the time oxidation of 0.3 mM ABTS (2, 2’- azinobis-3-ethyl--benzothiazoline--6-sulphonic acid) in detected in GP earlier than in LGP ( Fig. 2). The activity 50 mM sodium malonate buffer (pH 4.5). Catalase ac- levels of laccase, MnP and LiP in GP were higher than in tivity (EC 1.11.1.6) was assayed by measuring the de- LGP throughout the culture period; only AAO activity gradation H 2O2 (10 mM) in 100 mM potassium phos- was higher in LGP on days 4, 10, 12 and 22. phate buffer (pH 7.8). The rate of disappearance was On comparing different treatments, ligninolytic monitored at 240 nm (Cohen et al ., 1970). activity levels found in LP were approximately 10 times Enzyme assays were carried out at room temperature higher than those of LGP with the exception of MnP (25°C) using a Cari 50 Bio spectrophotometer (Varian, activity which was no longer detectable ( Fig. 3). Sugarland Tex.) during all incubation period. To start The activity pattern of catalase was similar in GP and measurements, 10 µL of extracellular protein sample was LGP, as it was high earlier in the culture period and added to 90 µL of reaction mixture (specific substrate decreased during incubation. Nevertheless, catalase dissolved in the buffer). One unit of enzyme activity was activity in LGP was 55-70% higher than in GP for the µ defined as 1 moL of formed per min per mg of first 8 days of culture. The highest activity levels of protein (U/mg of protein). Extracellular protein catalase occurred in LP (twice that of GP and LGP) 20 concentration was determined using Bradford’s method days after inoculation ( Fig. 4). (Bradford, 1976) using Bovine Serum Albumin (BSA) as The patterns of ligninolytic enzyme activities standard. All assays were done in triplicate. measured in LGP are shown in Fig. 2. The first activity A completely randomized design was applied to detected was that of AAO (0.018 U/mg of protein). After enzyme analyses. Treatments were tested by triplicate. 10 days, a second peak in AAO activity was detected and Independent variable was lignin and glucose and LiP activity increased, reaching its maximum at 12 days dependent variable the different enzymatic activities. (0.006 U/mg of protein). Laccase activity peaked immediately after AAO activity declined (at 14 days), 3. RESULTS whereas LiP remained fairly constant for 10 days. When LiP and laccase activity levels gradually decreased (day 3.1. Biomass Production 18), MnP activity increased and reached its maximum Different concentrations of lignin were tested for activity level (0.015 U/mg of protein) on the last day of their efficiency in stimulating biomass production. It was incubation (day 22). found that the highest mycelial growth was obtained by Table 1. Ratio of change in extracellular protein adding 1 mg of lignin/mL to the culture prepared with concentration vs . biomass production during 22 glucose and peptone (data not shown). Therefore, this days of L. edodes culture. concentration was selected for the analysis of enzyme Extracellular protein concentration/biomass activity. In the culture without glucose the mycelial bio------mass was not affected compared to with glucose even Time (days) GP LGP LP though it decreased 20% at the beginning and the end of 4 0.23 0.05 1.30 the culture period (Fig. 1A ). When the fungus was 6 0.45 0.17 1.12 cultivated in a medium containing Lignin and Glucose- 8 0.22 0.03 0.98 10 0.62 0.42 0.22 Peptone (LGP), mycelial growth was increased 45-70% 12 0.58 0.77 0.63 compared to that of the Glucose-Peptone medium 14 0.70 0.82 0.85 without lignin (GP), 14 days after inoculation ( Fig. 1B ). 16 1.25 1.09 0.12 When glucose was present in the medium, the 18 1.40 1.17 0.17 extracellular protein concentration increased during 20 1.05 1.05 0.50 fermentation. The highest levels were detected in LGP (ca. 22 1.11 1.16 0.53

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(A)

(B)

Fig. 1. The effect of culture conditions on the biomass of L. edodes with (A) lignin-peptone LP ( ●), glucose-peptone GP ( ■) and (B) lignin-glucose-peptone LGP ( ♦) added to the culture medium. Each point is the mean ± standard deviation. The experiment was carried out in independent

The levels of LiP and MnP activity detected in LGP In GP, LiP was the first enzyme for which activity were lower than those of GP for all the days sampled was detected (0.2 U/mg of protein 8 days after (Fig. 2). Figure 3 shows that over 14 days of incubation incubation), followed by AAO, MnP and laccase in LP medium, enzyme activity was not detectable activities that gradually increased, reaching their (except on day 8, when LiP activity was 0.1 U/mg of maximum level on day 10 (0.01, 0.01 and 0.08 U/mg protein). The highest LiP and AAO activities were detected at the same time (4.32 and 0.025 U/mg of of protein, respectively) ( Fig. 3 ). After that, LiP protein, respectively on day 16). After the activity of activity decreased and stayed constant and then both enzymes decreased, laccase activity increased to surprisingly peaked again on the last day of incubation 0.74 U/mg of protein on day 20. (0.25 U/mg of protein).

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Fig. 2. The effect of culture conditions on enzyme activity of L. edodes (laccase-LAC; aryl alcohol oxidase-AAO; lignin peroxidase- LiP; and manganese peroxidase-MnP) with lignin-glucose-peptone LGP ( ▲) and glucose-peptone GP ( ■) added to culture medium. Each point is the mean ± standard deviation

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Fig. 3. Ligninolytic enzymes activity (laccase-LAC; aryl alcohol oxidase-AAO; lignin peroxidase-LiP; and manganese peroxidase-MnP) of L. edodes on two culture medium with lignin-peptone LP ( ●) and glucose-peptone GP ( ■). Each point is the mean±standard deviation

Figure 2 and 4 show a decrease in catalase activity 2001; Ferrara et al ., 2002). A similar effect was observed once the activity of both peroxidases (MnP and LiP) in biomass production in the treatment containing Lignin increase in the GP and LGP treatments. A contrary effect and Glucose (LGP). Nevertheless, when glucose was was found in LP cultures ( Fig. 3-4): Catalase activity absent from the medium supplied with Lignin (LP), was gradually increased to higher levels and MnP biomass production was not different from that of the activity remained undetected. In the same treatment cultures without lignin (GP) ( Fig. 1A ). (LP), LiP activity peaked before catalase reached its In general, biomass production was similar in the maximum activity level and there was a slight increase in GP and LP cultures, while the enhancement of fungal LiP when catalase activity decreased ( Fig. 3-4). growth was observed when lignin and glucose were added to the medium. This indicates that a simple 4. DISCUSSION carbon source combined with lignin had a positive effect on the mycelial biomass. It was not possible to 4.1. Biomass Production know whether L. edodes used lignin as the carbon source It has been established that lignin derived phenols or if lignin was simply a growth promoter. However, and polymeric lignin play a role in stimulating growth in there was an increase of biomass and that is probably a white-rot fungi, including L. edodes (Beltrán-García et al ., result of the solubilization of lignin monomers.

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Fig. 4. Catalase activity of L. edodes on lignin-glucose-peptone LGP ( ♦), glucose-peptone GP ( ■) and lignin-peptone LP ( ●) culture medium. Each point is the mean±standard deviation

4.2. Enzymatic Activity potential than MnP and cleaves bonds between the nonphenolic subunits that are the major component of lignin Some experiments have demonstrated that when (Faison and Kirk, 1983). In the present study, LiP activity added to the culture medium, polymeric lignin and was found to be enhanced before MnP activity in GP different aromatic and phenolic molecules (derived from medium and the highest values were recorded for LP 16 or related to lignin) enhance the activity and transcript days after incubation. levels of laccase in different fungi (Tychanowicz et al ., Catalase activity was measured because of the 2004; Covallazzi et al ., 2005; Salmones and Mata, importance of H 2O2 in the process of lignin degradation 2005). Additionally, it has been proposed that laccase and catalase might help to protect this fungus against induction stops when the glucose has been consumed and H2O2, which is a substrate for peroxidases, but H 2O2 when compounds have been solvated or liberated during could permeate the fungal membrane and, in the lignin degradation (Ha et al ., 2001; Moldes et al ., 2004). presence of metal ions, damage cellular structures, The results presented in Fig. 2 do not agree with those including DNA (Doudican et al ., 2005). Faison and Kirk mentioned above because laccase activity was lower in (1983) demonstrated that the addition of catalase to LGP than in GP, in addition to being detected later. cultures of Phanerochaete chrysosporium inhibits lignin Nevertheless, in LP medium, laccase activity was degradation and this could be related to the results enhanced during the latter part of the incubation period. reported in the present study. In LGP cultures, this activity would probably increase In L. edodes , it was found that ligninolytic activity after glucose has been depleted. was stimulated earlier by adding easily metabolized It has been proposed that LiP is the enzyme responsible carbohydrates (Leatham, 1985), so it is possible that, for the initial attack of lignin because it has higher redox relative to the LP medium, the GP medium tested here

Science Publications 376 AJABS Citlalli Harris-Valle et al . / American Journal of Agricultural and Biological Sciences 9 (3): 369-378, 2014 exerts similar effect on enzyme activity. Nevertheless, in 5. CONCLUSION LGP the relationship between the carbon source and the stimulation of enzyme activity was not clear, probably The present results show that lignin and glucose because the presence of lignin modified the environment together supplemented in a liquid culture medium of L. and the enzymes activity pattern was more complex. It is edodes enhanced biomass and modified the activity possible that during incubation, lignin was degraded and pattern of ligninolytic enzymes, but did not enhance liberated several soluble compounds of low molecular weight that modulated the lignin degradation system, enzyme activity, compared to media without lignin and which is supported by this work. This effect was without glucose. Therefore we propose that this suggested by Faison and Kirk (1985) in solid cultures of polymer exerts an influence as great as that of a simple P. chrysosporium . They found that lignin degradation can carbon source on the activity profile and biomass be induced by several substrates (or products) including production of ligninolytic enzymes. lignin monomers and oligomers. In addition, when white rot fungus is cultivated in lignocellulosic substrates, 6. ACKNOWLEDGEMENT ligninolytic activity is a secondary metabolic event. Lignin degrading enzymes have different profiles and activity The researchers thank Dr. Hiroshi Ogura for his levels depending on the culture system (Ha et al ., 2001; valuable suggestions regarding ligninolysis and on the Ohga and Royse, 2001). manuscript. A. Gutierrez for his assistance. Citlali Although it is thought that white-rot fungus must de- Harris-Valle thanks CONACYT for her scholarship. grade an alternate substrate in order to carry out lignin depolymerization, some authors have found that organ- 7. REFERENCES isms hydrolyze polysaccharides at sufficient rates for optimal growth and maintenance of ligninolytic activity Aro, N., T. Pakula and M. Penttila, 2005. Transcriptional (Leatham, 1985; Vane et al ., 2003). During regulation of plant cell wall degradation by delignification, cellulose is broken down simultaneously filamentous fungi. FEMS Microbiol. Rev., 29: 719- and hemicellulose is an energy source for white rot fungi, 739. PMID: 16102600 since lignin alone apparently cannot serve as a growth Beltrán-García, M.J., A. Orozco, I. Samayoa and T. substrate (Elisashvili and Kachlishvili, 2009). Those findings could explain, in part, our data for the LP Ogura, 2001. Lignin degradation products from corn treatment. Mycelial growth was similar to that of GP stalks enhance notable the radial growth of throughout the culture, there was no MnP activity basidiomycete mushroom mycelia. Rev. Soc. Quím. detected, maximum catalase activity was recorded at the Méx, 45: 77-81. end of incubation and LiP was low. Laccase and AAO Boer, C.G., L. Obici, C.G. Marques de Souza and R.M. activity were detected earlier in the culture, though high Peralta, 2004. Decolorization of synthetic dyes by levels were reached 14 days after inoculation. solid state cultures of Lentinula (Lentinus) edodes Basidiomycetes growing in toxic environments producing manganese peroxidase as the main (phenols, resins and other wood components), with ligninolytic enzyme. Biores. Technol., 94: 107-112. limited nutrient availability can successfully compete PMID: 15158501 with other organisms for substrate. Lignin Bradford, M., 1976. A rapid and sensitive method for the depolymerization is in part achieved by a free radical quantitation of microgram quantities of protein mechanism based upon the radical generated by utilizing the principle of protein-dye binding. Anal. peroxidase complexes in the presence of Hydrogen Biochem., 72: 248-254. DOI: 10.1016/0003- Peroxide (H 2O2 produced by gliox-al oxidases and 2697(76)90527-3 glucose oxidase and aryl alcohol oxidase activities). Busse, N., D. Wagner, M. Kraume and P. Czermak, The fungal enzymes then solubilize lignin, the lignin 2013. Reaction kinetics of versatile peroxidase for degradation products (including phenolic monomers) diffuse and the mycelial growth of the fungus the degradation of lignin compounds. Am. J. accelerates. As a result, more degradation takes place. Biochem. Biotechnol., 9: 365-394. DOI: 10.38- We feel this is an important tactic for fungi to secure 44/ajbbsp.2013.365.394 their niche on a substrate before their competitors can. Cohen, G., D. Dembiec and J. Marcus, 1970. With results obtained here, our next study will focus on Measurement of catalase activity in tissue extracts. whether lignin can be used as carbon source and, if it Anal. Biochem, 34: 30-38. DOI: 10.1016/0003- can, whether that would affect lignin depolymerization. 2697(70)90083-7

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